Activated Rho GTPases signal to numerous intracellular proteins to dynamically control vital cellular processes, including remodeling of the actin cytoskeleton, membrane trafficking, transcriptional regulation, cell growth, and development. The activation of Rho GTPases is catalyzed by Rho GEFs (guanine nucleotide exchange factors) in response to a variety of extracellular signals. This process is tightly controlled and spatially focused within cells, and abnormal regulation of several Rho GTPases is implicated in human cancers and other diseases. The long-term goal of this research is to more clearly define molecular mechanisms of Rho GTPase regulation in order to increase our understanding of Rho GTPase function in normal and pathological states and to improve our capacity to treat human diseases that stem from aberrant Rho GTPase signaling. The overall objective of this proposal is to develop a workflow comprised of an integrated set of assays for rapid identification and validation of compounds that can specifically and directly inhibit guanine nucleotide exchange and be used as probes of Rho GTPase function in cells. The goals of this proposal will be accomplished through three aims. In Specific Aim 1, a fluorescence-based nucleotide exchange assay will be optimized for high-throughput screening. In Specific Aim 2, high-throughput screening of diverse compound libraries will be implemented to identify inhibitors of P-Rex2-catalyzed activation of Rac1 and combined with structure similarity searching to improve the number and potency of initial hits. In Specific Aim 3, bona fide in vitro inhibition will be confirmed with a conventional radioactive-based exchange assay, and a series of in vitro and cell-based secondary assays will be developed to define the selectivity, cytotoxicity and biological activity of confirmed hits. The protocols developed here for Rac1 will be generally applicable to all Rho GTPases and are expected to yield lead compounds for generation of potent and selective probes of Rho GTPase function. These compounds may also serve as potential lead compounds for drug development.